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. 2019 Sep:26:101302.
doi: 10.1016/j.redox.2019.101302. Epub 2019 Aug 15.

Serum response factor (SRF) promotes ROS generation and hepatic stellate cell activation by epigenetically stimulating NCF1/2 transcription

Ming Kong  1 , Xuyang Chen  1 , Fangqiao Lv  2 , Haozhen Ren  3 , Zhiwen Fan  3 , Hao Qin  1 , Liming Yu  1 , Xiaolei Shi  4 , Yong Xu  5
Affiliations

Serum response factor (SRF) promotes ROS generation and hepatic stellate cell activation by epigenetically stimulating NCF1/2 transcription

Ming Kong et al. Redox Biol. 2019 Sep.

Abstract

Activation of hepatic stellate cells (HSC) is a hallmark event in liver fibrosis. Accumulation of reactive oxygen species (ROS) serves as a driving force for HSC activation. The regulatory subunits of the NOX complex, NCF1 (p47phox) and NCF2 (p67phox), are up-regulated during HSC activation contributing to ROS production and liver fibrosis. The transcriptional mechanism underlying NCF1/2 up-regulation is not clear. In the present study we investigated the role of serum response factor (SRF) in HSC activation focusing on the transcriptional regulation of NCF1/2. We report that compared to wild type littermates HSC-conditional SRF knockout (CKO) mice exhibited a mortified phenotype of liver fibrosis induced by thioacetamide (TAA) injection or feeding with a methionine-and-choline deficient diet (MCD). More importantly, SRF deletion attenuated ROS levels in HSCs in vivo. Similarly, SRF knockdown in cultured HSCs suppressed ROS production in vitro. Further analysis revealed that SRF deficiency resulted in repression of NCF1/NCF2 expression. Mechanistically, SRF regulated epigenetic transcriptional activation of NCF1/NCF2 by interacting with and recruiting the histone acetyltransferase KAT8 during HSC activation. In conclusion, we propose that SRF integrates transcriptional activation of NCF1/NCF2 and ROS production to promote liver fibrosis.

Keywords: Hepatic stellate cell; Liver fibrosis; Neutrophil cytosolic factor; Reactive oxygen species; Serum response factor; Transcriptional regulation.

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Figures

Fig. 1
Fig. 1
HSC-specific SRF deficiency attenuates TAA-induced liver fibrosis in mice. WT and CKO mice were injected with TAA to induce liver fibrosis as described in Methods. (A, B) Plasma ALT and AST levels. (C) Pro-fibrogenic gene expression was measured by qPCR. (D, E) Picrosirus red and Masson's trichrome staining. (F) Hepatic hydroxyl proline levels. N = 5 mice for each group. Error bars represent SD (*p < 0.05, 2-tailed student's t-test).
Fig. 2
Fig. 2
HSC-specific SRF deficiency attenuates MCD-induced liver fibrosis in mice. WT and CKO mice were fed with a MCD diet to induce liver fibrosis as described in Methods. (A, B) Plasma ALT and AST levels. (C) Pro-fibrogenic gene expression was measured by qPCR. (D, E) Picrosirus red and Masson's trichrome staining. (F) Hepatic hydroxyl proline levels. N = 5 mice for each group. Error bars represent SD (*p < 0.05, 2-tailed student's t-test).
Fig. 3
Fig. 3
SRF regulates ROS levels in hepatic stellate cells. (A) Liver fibrosis was induced by TAA in WT and CKO mice as described in Methods. Cryosections were co-stained with DHE and anti-desmin. N = 5 mice for each group. Error bars represent SD (*p < 0.05, 2-tailed student's t-test). (B) Liver fibrosis was induced by MCD feeding in WT and CKO mice as described in Methods. Cryosections were co-stained with DHE and anti-desmin. N = 5 mice for each group. Error bars represent SD (*p < 0.05, 2-tailed student's t-test). (C) LX-2 cells were transfected with siRNA targeting SRF or scrambled siRNA (SCR) followed by staining with DHE and DCFH. Data represent averages of three independent experiments and error bars represent SEM (*p < 0.05, 2-tailed student's t-test). (D) Primary mouse HSCs were transfected with siRNA targeting SRF or SCR followed by staining with DHE and DCFH. Data represent averages of three independent experiments and error bars represent SEM (*p < 0.05, 2-tailed student's t-test).
Fig. 4
Fig. 4
SRF activates NCF1/NCF2 transcription in hepatic stellate cells. (A) WT and CKO mice were induced to develop liver fibrosis by TAA injection. Hepatic levels of NCF1 and NCF2 were examined by qPCR. (B) WT and CKO mice were induced to develop liver fibrosis by MCD diet. Hepatic levels of NCF1 and NCF2 were examined by qPCR. (C) LX-2 cells were transfected with siRNA targeting SRF or SCR. Expression levels of NCF1 and NCF2 were examined by qPCR. (D) Primary mouse HSCs were transfected with siRNA targeting SRF or SCR. Expression levels of NCF1 and NCF2 were examined by qPCR. (E) Wild type (WT) or mutant (MT) NCF1 promoter-luciferase construct was transfected into HEK293 and LX-2 cells with or without SRF. Luciferase activities were normalized by GFP fluorescence and protein concentration. (F) Wild type (WT) or mutant (MT) NCF2 promoter-luciferase construct was transfected into HEK293 and LX-2 cells with or without SRF. Luciferase activities were normalized by GFP fluorescence and protein concentration. (G) Nuclear lysates were extracted from LX-2 cells and ChIP assays were performed with anti-SRF or IgG. Data represent averages of three independent experiments and error bars represent SEM (*p < 0.05, 2-tailed student's t-test).
Fig. 5
Fig. 5
SRF recruits KAT8 to activate NCF1/NCF2 transcription. (A-F) LX-2 cells were transfected with siRNA targeting SRF or SCR. ChIP assays were performed with indicated antibodies. (G) HA-tagged KAT8 and GFP-tagged SRF were transfected into HEK293 cells. Immunoprecipitation was performed with anti-HA or anti-GFP. (H) Nuclear lysates were extracted from LX-2 cells. Immunoprecipitation was performed with indicated antibodies. (I) Nuclear lysates were extracted from LX-2 cells and Re-ChIP assays were performed with indicated antibodies. (J) Primary mouse HSCs were isolated and nuclear lysates were extracted at 7d after activation. Re-ChIP assays were performed with indicated antibodies. Data represent averages of three independent experiments and error bars represent SEM (*p < 0.05, 2-tailed student's t-test).
Fig. 6
Fig. 6
KAT8 is essential for HSC activation by regulating NCF1/NCF2 transcription. (A-C) LX-2 cells were transfected with siRNA targeting KAT8 or SCR. Gene expression levels were examined by qPCR and Western. ChIP assays were performed with anti-acetyl H4K16. (D-F) Primary mouse HSCs were transfected with siRNA targeting KAT8 or SCR. Gene expression levels were examined by qPCR and Western. ChIP assays were performed with anti-acetyl H4K16. Data represent averages of three independent experiments and error bars represent SEM (*p < 0.05, 2-tailed student's t-test). (G) A schematic model.
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